Human Intestinal Epithelial Caco-2 Research Articles

The surfactant co-formulant POEA in the glyphosate-based herbicide RangerPro but not glyphosate alone causes necrosis in Caco-2 and HepG2 human cell lines and ER stress in the ToxTracker assay.

The toxicity of co-formulants present in glyphosate-based herbicides (GBHs) has been widely discussed leading to the European Union banning the polyoxyethylene tallow amine (POEA). We identified the most commonly used POEA, known as POE-15 tallow amine (POE-15), in the widely used US GBH RangerPro. Cytotoxicity assays using human intestinal epithelial Caco-2 and hepatocyte HepG2 cell lines showed that RangerPro and POE-15 are far more cytotoxic than glyphosate alone. RangerPro and POE-15 but not glyphosate caused cell necrosis in both cell lines, and that glyphosate and RangerPro but not POE-15 caused oxidative stress in HepG2 cells. We further tested these pesticide ingredients in the ToxTracker assay, a system used to evaluate a compound's carcinogenic potential, to assess their capability for inducing DNA damage, oxidative stress and an unfolded protein response (endoplasmic reticulum, ER stress). RangerPro and POE-15 but not glyphosate gave rise to ER stress. We conclude that the toxicity resulting from RangerPro exposure is thus multifactorial involving ER stress caused by POE-15 along with oxidative stress caused by glyphosate. Our observations reinforce the need to test both co-formulants and active ingredients of commercial pesticides to inform the enactment of more appropriate regulation and thus better public and environmental protection.

ADENOSINE A3 RECEPTOR INTERACTS WITH GASDERMIN D TO MODULATE INTESTINAL EPITHELIAL CELL PYROPTOSIS IN ULCERATIVE COLITIS

Abstract Background Adenosine A3 receptor (A3AR) plays a role in intestinal inflammation, but little is known about its mechnisms in intestinal inflammation such as ulcerative colitis (UC). Pyroptosis, characterized by Gasdermin D (GSDMD) activation, is implicated in the pathogenesis of UC. We investigated the role of A3AR in GSDMD-mediated pyroptosis in UC and its underlying molecular mechanisms. Methods The expression of A3AR in colonic mucosa of patients with UC were examined. A3AR agonist was used to study the role of A3AR in ex vivo colonic explants of UC patients. In addition, human intestinal epithelial cells Caco-2 were used to further verify the effect of A3AR on pyroptosis induced by LPS+ATP. RT-qPCR and western blotting were used to detect the expression levels of pyroptosis-associated factors including NLRP3, caspase-1, gasdermin-D N-terminal domain (GSDMD-NT), IL-1β and IL-18 in colonic tissues and Caco-2 cells. Immunofluorescence was used to detect the protein expression in tissues and cells. Enzyme-linked immunosorbent assay was used to determine the levels of IL-1β and IL-18 in tissue and cell culture supernatants. Lactate dehydrogenase (LDH) release assay and propidium iodide (PI) staining were used to measure cell pyroptosis. Molecular interactions beween A3AR and GSDMD were investigated using Co-IP and GST pull-down assays. Results A3AR expression was significantly reduced in colonic mucosa of patients with active UC, and colonic epithelial cells were the main cell subpopulation with down-regulated A3AR expression. Pyroptosis-associated factors, including Caspase-1, NLRP3, and GSDMD-NT, were upregulated in UC colonic tissues. The expression of A3AR and GSDMD-NT was negatively correlated. A3AR agonist reduced the production of cytokines (IL-1β and IL-18) and attenuates the expression levels of NLRP3 and GSDMD-NT in the colonic tissues of patients with UC. Furthermore, A3AR overexpression alleviated pyroptosis with reduced LDH release, PI-stained cell number and decreased expressions of GSDMD-NT, NLRP3, caspase-1, IL-1β, and IL-18 in the LPS+ATP-stimulated Caco-2 cells, whereas the opposite occurred in cells treated with small interfeing RNA (siRNA) targeting A3AR. Knockdown of GSDMD in Caco-2 cells significantly blocked the effects of A3AR overexpression or down-regulation on pyroptosis, suggesting that A3AR acts through the GSDMD-mediated pyroptosis pathway. Co-IP and GST pull-down assays showed that A3AR interacted with GSDMD. Conclusion A3AR modulates intestinal inflammation in UC through GSDMD-mediated intestinal epithelial cell pyroptosis. We propose a novel mechanism by which A3AR-GSDMD interaction affects UC through pyroptosis, suggesting that A3AR is a potential target for the treatment of UC.

Virulence characterization and comparative genomics of Listeria monocytogenes sequence type 155 strains

BackgroundListeria (L.) monocytogenes strains show a high diversity regarding stress tolerance and virulence potential. Genome studies have mainly focused on specific sequence types (STs) predominantly associated with either food or human listeriosis. This study focused on the prevalent ST155, showing equal distribution among clinical and food isolates. We evaluated the virulence potential of 20 ST155 strains and performed comparative genomic analysis of 130 ST155 strains isolated from food, food processing environments and human listeriosis cases in different countries and years.ResultsThe in vitro virulence assays using human intestinal epithelial Caco2 and hepatocytic HEPG2 cells showed an impaired virulence phenotype for six of the 20 selected ST155 strains. Genome analysis revealed no distinct clustering of strains from the same source category (food, food processing environment, and clinical isolates). All strains harbored an intact inlA and inlB locus, except four strains, which had an internal deletion in the inlA gene. All strains harbored LIPI-1, but prfA was present in a longer variant in six strains, all showing impaired virulence. The longer PrfA variant resulted in lower expression of inlA, inlB, and prfA, and no expression of hly and actA.Regarding stress-related gene content, SSI-1 was present, whereas qacH was absent in all strains. 34.6% of the strains harbored a plasmid. All but one ST155 plasmids showed high conservation and harbored cadA2, bcrABC, and a triphenylmethane reductase.ConclusionsThis study contributes to an enhanced understanding of L. monocytogenes ST155 strains, being equally distributed among isolates from humans, food, and food processing environments. The conservation of the present genetic traits and the absence of unique inherent genetic features makes these types of STs especially interesting since they are apparently equally adapted to the conditions in food processing environments, as well as in food as to the human host environment. However, a ST155-specific mutation resulting in a longer PrfA variant impaired the virulence potential of several ST155 strains.

Palmitic acid damages gut epithelium integrity and initiates inflammatory cytokine production

The mechanisms leading to the low-grade inflammation observed during obesity are not fully understood. Seeking the initiating events, we tested the hypothesis that the intestine could be damaged by repeated lipid supply and therefore participate in inflammation. In mice, 1–5 palm oil gavages increased intestinal permeability via decreased expression and mislocalization of junctional proteins at the cell–cell contacts; altered the intestinal bacterial species by decreasing the abundance of Akkermansia muciniphila, segmented filamentous bacteria, and Clostridium leptum; and increased inflammatory cytokine expression. This was further studied in human intestinal epithelial Caco-2/TC7 cells using the two main components of palm oil, i.e., palmitic and oleic acid. Saturated palmitic acid impaired paracellular permeability and junctional protein localization, and induced inflammatory cytokine expression in the cells, but unsaturated oleic acid did not. Inhibiting de novo ceramide synthesis prevented part of these effects. Altogether, our data show that short exposure to palm oil or palmitic acid induces intestinal dysfunctions targeting barrier integrity and inflammation. Excessive palm oil consumption could be an early player in the gut alterations observed in metabolic diseases.

Overproduction, purification, and characterization of nanosized polyphosphate bodies from Synechococcus sp. PCC 7002

BackgroundInorganic polyphosphate bodies (PPB) have recently been linked to a variety of functions in mammalian cells. To improve the yield of PPB from Synechococcus sp. PCC 7002 and characterize its form, in this study, a recombinant plasmid containing a polyphosphate kinase (ppk) gene was generated and transformed into Synechococcus sp. PCC 7002.ResultsPPB separated by Sephadex G-100 was characterized and added to polarized human intestinal epithelial (Caco-2) cells, and the absorption effect was assessed. The ppk gene was stably expressed by induction with 1 μM nickel, and the resulting PPB yield from Synechococcus sp. PCC 7002 cells increased by 89.66%. Transmission electron microscopy and dynamic light scattering analyses showed that PPB from these cells were nanosized, ranging from a few to approximately 100 nanometres in diameter. PPB can be taken up by Caco-2 cells and are mainly distributed around lipid droplets.ConclusionsWe determined that PPB can be overproduced in Synechococcus sp. PCC 7002 and that the resulting PPB were well absorbed by Caco-2 cells. Microalgae provide a promising “cell factory” for PPB production.

In-vitro toxicity of carbon nanotube/polylysine colloids to colon cancer cells.

Single-walled carbon nanotubes (SWCNTs) are thoroughly purified and dispersed in an aqueous solution of high molecular weight poly-L-lysine (pLlys). Human intestinal epithelial Caco-2/TC7 cells are incubated with the SWCNT dispersions in pLlys, and their effects on cell viability are studied by image flow cytometry. No significant changes are observed in the cell culture wells up to pLlys concentrations of 10 μg ml-1. However, high mortality is detected at pLlys concentrations of 100 μg ml-1. The presence of oxygen-free SWCNTs does not modify the effects of pLlys on cell cultures at any of the tested concentrations (≤1 μg ml-1). In addition, SWCNTs having an 8 wt.% of surface oxygen are tested with identical results. Thus, purified SWCNTs, even bearing oxygen functional groups, act as inert particles in the cell culture medium. This result supports the applicability of SWCNTs as carriers in pharmacological formulations against digestive tract diseases.

P048. Cigarette smoke impairs intestinal epithelial cell polarity development and morphogenesis

Background: Cigarette smoking is emerging as an important lifestylefactor that affects intestinal function. Epidemiological studies indicated that cigarette smoking influences the course of inflammatory bowel disease and is a risk factor for colorectal cancer andintestinal polyp development. The mechanism via which cigarette smoke exerts its effect on the intestinal epithelium is unclear. These may involve indirect effects via alterations in the immune system or direct effects of cigarette smoke on the intestinal epithelium. Little is known about these direct effects of cigarette smoke on intestinal epithelial cells. Here we present an in-vitro study of these direct effects Methods: Human intestinal epithelial Caco-2 cell-matrigel-embedded spheriods were grown for 7 days in the presence of, or pre treated with, cigarette smoke extract (CSE). For pre treatment cell were incubated with CSE for 24 hours, and subsequently plated out in fresh in3D culture. Intestinal epithelial barrier function was measured witha FITC-dextran leakage assay. Cell polarity and morphogenesis were assessed with fluorescently labelled polarity markers Results: We found that CSE did not affect the polarity and barrier function of pre established lumen-forming Caco-2 spheroids.However, when exposed to CSE during the process of 3D morphogenesis,cells showed impaired growth and impaired de novo development of apical-basal cell polarity and tight junctions, resulting in defective self-assembly into single lumen-forming spheroids. This effect was dose-dependent and this phenotype could be induced with pre incubation with CSE as well. However, the administration of the glutathione precursor N-acetyl-L-cysteine (NAC) completely neutralised the inhibitory effects of CSE. The mechanisms by which NAC rescued the phenotype were independent of its capacity to induce glutathione production Conclusions: Our data demonstrate that CSE has direct and persistentinhibitory effects on intestinal epithelial cell polarity developmentand morphogenesis, and provide insight into the cell biologicalmechanisms. NAC supplementation was identified as a protectivestrategy for smoke-induced epithelial dysfunction

Effects of hydrogen-rich medium on lipopolysaccharide-induced intestinal epithelial barrier dysfunction of human colon carcinoma cells

To investigate the effects of hydrogen-rich medium on lipopolysaccharide (LPS)-induced intestinal epithelial barrier dysfunction of human intestinal epithelial (Caco2) cells. Caco2 cells (passages 28-35) were purchased from the Cell Bank of the Shanghai Institute of Cell Biology, Chinese Academy of Sciences in Shanghai, China, and they were cultured in Dulbecco minimum essential medium (DMEM) containing 20% fetal bovine serum. These cells were randomly divided into four groups: control group (group A), hydrogen-rich medium group (group B), LPS group (group C) and LPS + hydrogen-rich medium group (group D). Cells were cultured with normal medium in group A and group C or with hydrogen-rich medium in group B and group D. Meanwhile, 1 g/L LPS was simultaneously added into group C and group D, while an equivalent volume of normal saline was added into group A and group B instead. In vitro intestinal epithelial models were reproduced with monolayer filter-grown Caco2 and intestinal epithelium. The trans-epithelial electrical resistance (TEER) in models of each group was measured at different incubation times (0, 3, 6, 12, 24 and 48 hours). Cell viability and cytotoxicity were assessed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and lactate dehydrogenase (LDH) release assay, respectively, after incubation for 24 hours. The expression levels of claudin-1 and occludin were respectively determined at 6, 12 and 24 hours of incubation by Western Blot assay. The morphological structure of claudin-1 and occludin was respectively observed after incubation for 24 hours with immunofluorescence staining. There was no statistical significance in variables between group A and group B. Compared with group A, it was shown that TEER was time-dependently decreased in groups C and D after 6 hours. Compared with group C, TEER in group D was increased after 6 hours. Compared with group A, the cell viability was significantly reduced in group C [(67.2±7.9)% vs. (100.0±0.0)%, P < 0.05] and cell injury was obvious [LDH release rate: (38.5±2.1)% vs. (1.2±0.3)%, P < 0.05]; the expression levels of claudin-1 and occludin at 6, 12, 24 hours were significantly down-regulated [claudin-1 (gray value): 0.351±0.079, 0.272±0.075, 0.190±0.049 vs. 0.518±0.030; occludin (gray value): 0.416±0.044, 0.290±0.062, 0.226±0.019 vs. 0.602±0.038, all P < 0.05], and the structure of claudin-1 and occludin were profoundly disrupted. Compared with group C, it was shown that the cell viability was significantly increased in group D [(88.8±7.4)% vs. (67.2±7.9)%, P < 0.05] and cell injury was significantly abated [LDH release rate: (16.4±4.3)% vs. (38.5±2.1)%, P < 0.05]; the expression levels of claudin-1 and occludin were significantly up-regulated at 24 hours [claudin-1 (gray value): 0.428±0.046 vs. 0.190±0.049, occludin (gray value): 0.466±0.071 vs. 0.226±0.019, both P < 0.05]; the disrupted structures of claudin-1 and occludin were partially recovered. Hydrogen-rich medium can effectively attenuate LPS-induced dysfunction of intestinal epithelial barrier in human Caco2 cells by ameliorating cell viability as well as regulating claudin-1 and occludin expression and structure.

P746. Anti-inflammatory properties of Faecalibacterium prausnitzii in the absence and presence of cigarette smoke using a novel epithelium-gut microbe coculture system

Background: Inflammatory bowel disease (IBD) is characterised bya recurrent inflammation of the gut epithelium. Although the pathogenesis is still largely unknown, different factors contribute to the disease, including genetic and environmental factors. Cigarette smoking is the most-studied environmental factor associated with IBD. Moreover, gut microbiota play a central role in the pathogenes is ofIBD. In this study we aimed to investigate the effects of host-microbe interactions on the gut epithelium with a potentially beneficial bacterium,Faecalibacterium prausnitzii, in a recently established coculture system for anaerobic bacteria and oxygen-requiring humanintestinal epithelial (Caco-2) cells. Hence, we analysed the effect of an inflammatory cytokine (TNF-α) and cigarette smoke extract(CSE) on the growth of F. prausnitzii and expression of inflammatory markers in Caco-2 cells. Methods: F. prausnitzii and human Caco-2 cells were cocultured in the ‘Human oxygen Bacteria anaerobic’ (HoxBan) system and exposed to TNF-α and/or CSE. Bacterial growth was monitored visually, and expression of the inflammatory marker iNOS and oxidative stress marker HO-1 were determined by quantitative PCR. Results: F. prausnitzii grew well in the HoxBan coculturing system in the presence of TNF-α or with added CSE, with no observable difference to control conditions. F. prausnitzii strongly suppressed iNOS expression, both in TNF-α treated and control Caco-2 cells.CSE did not induce iNOS or HO-1 expression in Caco-2 cells,but F. prausnitzii strongly suppressed iNOS expression both in the absence and presence of CSE, with similar trends observed for HO-1. Conclusions: We conclude that F. prausnitzii produces potent antiinflammatoryfactors, which is independent of cigarette smoking.F. prausnitzii and its secreted products are therefore interesting targetsfor the treatment of intestinal inflammation, in particular forIBD

Transport Mechanism of Intestinal Absorption of μ Opioid Recept or Agonists and Contribution of P-Glycoprotein in Rats and Human Intestinal Epithelial Caco-2

Abstract 1.1. Introduction: The μ opioid receptor agonists, morphine and loperamide, are widely used orally and are suggested to be P-glycoprotein (P-gp) substrates. P-gp is expressed in the brain, intestine, and various tissues in human and rats. In the intestine, P-gp limits the absorption of certain drugs such as opioids; however, the underlying mechanism has not been elucidated. The aim of the present study was to examine the intestinal transport characteristics of morphine and loperamide and the role of P-gp in their transport process. 1.2. Method: Transcellular transport studies were conducted using isolated rat intestinal tissue mounted in an Ussing-type chamber. Bidirectional permeability and inhibition transport studies were performed using Caco-2 cell lines. The intestinal absorption was examined by an in situ closed-loop method in rats. 1.3. Results: Loperamide showed secretory transport across rat intestinal tissue and Caco-2 cells, and P-gp substrates cyclosporine A and rhodamine 123 inhibited this transport. In the intestinal loop experiment in rats, the accumulation of loperamide in the intestinal tissue increased upon adding cyclosporine A and rhodamine 123. In contrast, morphine showed no directional transport and P-gp inhibitory effects across rat intestinal tissue. In Caco-2 cells, morphine transport was found to be secretory-directed and this transport was inhibited by cyclosporine A and rhodamine 123, but to a much lesser extent than that of loperamide. Morphine disappearance and accumulation were unaffected upon the addition of cyclosporine A and rhodamine 123. 1.4. Conclusion: These results suggest that P-gp contributes significantly to the secretory transport of loperamide but negligibly to that of morphine in the small intestine. In conclusion, intestinal transport of both morphine and loperamide is found to be secretory-directed. P-gp partially contributes to this secretory-directed transport. Thus, P-gp is prominent in loperamide rather than morphine transport.

Activation of cofilin and its relation with distribution of tight junction protein zonula occludens 1 in hypoxic human intestinal epithelial cells

To study the effect of hypoxia on cofilin activation in intestinal epithelial cells and its relation with distribution of tight junction protein zonula occludens 1 (ZO-1). The human intestinal epithelial cell line Caco-2 was used to reproduce monolayer cells. The monolayer-cell specimens were divided into control group (no treatment), hypoxic group ( exposed to hypoxia), and normoxic group (exposed to normoxia) according to the random number table. Western blotting was used to detect the protein expressions of cofilin and phosphorylatedl cofilin (p-cofilin) of cells in normoxic group and hypoxic group exposed to normoxia or hypoxia for 1, 2, 6, 12, and 24 h and control group, with 9 samples in control group and 9 samples at each time point in the other two groups. The other monolayer-cell specimens were divided into hypoxic group (exposed to hypoxia) and control group (no treatment) according to the random number table. Cells in hypoxic group exposed to hypoxia for 1, 2, 6, 12, and 24 h and control group were obtained. Morphology and distribution of F-actin was observd with laser scanning confocal microscopy, the ratio of F-actin to G-actin was determined by fluorescence method, and distribution of ZO-l and cellular morphology were observed with laser scanning confocal microscopy. The sample number of last 3 experiments was respectively 3, 6, and 3 in both hypoxic group (at each time point) and control group. Data were processed with paired ttest, analysis of variance of repeated measurement, and LSD-t test. The protein expressions of cofilin and p-cofilin of cells between normoxic group exposed to normoxia for 1 to 24 h and control group showed no significant changes (with values from -0.385 to 1.701, t(p-cofilin)values from 0. 040 to 1.538, P values above 0.05). There were no obvious differences in protein expressions of en filmn of cells between hypoxic group exposed to hypoxia for 1 to 24 h and control group ( with values from 1.032 to 2.390, P values above 0.05). Compared with that in control group, the protein expressions of p-cofilin of cells were greatly reduced in hypoxic group exposed to hypoxia for 1 to 24 h (with values from 4.563 to 22.678, P values below 0.01), especially exposed to hypoxia for 24 h. The protein expressions of cofilin of cells between normoxic group and hypoxic group at each time point were close ( with t values from -0.904 to 1.433, P values above 0.05). In hypoxic group, the protein expressions of p-cofilin of cells exposed to hypoxia for 1, 2, 6, 12, and 24 h were 0.87 +/- 08, 0.780 .05, 0.89 +/- 0.07, 0.68+0. 07, and 0.57 +/- 0.06, respectively, significantly lower than those in normoxic group (0.90 +/- 0.07, 0.97 +/- 0.06, 1.00 +/- 0.06, 1.00 +/- 0.05, and 0.99 +/- 0.05, with t values from 3.193 to 16.434, P values below 0.01). In control group, F-actin in the cytoplasm was abundant, most of it was in bunches. The trend of F-actin was disorderly in hypoxic group from being exposed to hypoxia for 1 h, shortened in length or even dissipated. The ratios of F-actin to G-actin of cells in hypoxic group exposed to hypoxia for 12 and 24 h (0.89 +/- 0.12 and 0.84 +/- 0.19) were obviously decreased as compared with that in control group (1. 00, with t values respectively 3. 622 and 3. 577, P values below 0.01). There were no obvious differences in the ratios of F-actin to G-actin of cells between hypoxic group exposed to hypoxia for 1, 2, and 6 h and control group ( with values from 0.447 to 1.526, P values above 0.05). In control group, cells were compact in arrangement, and ZO-1 was distributed continuously along the cytomnembrane. From being exposed to hypoxia for 2 h, cells became irregular in shape in hypoxic group. ZO-1 was distributed in discontinuous fashion along the cytomembrane with breakage in hypoxic group exposed to hypoxia for 24 h. Hypoxia may cause the disorder of dynamic balance between F-actin and G-actin by inducing cofilin activation, which in turn leads to the changes in distribution of tight junction protein ZO-1 in intestinal epithelial cells.

Listeria monocytogenes isolated from food samples from a Romanian black market show distinct virulence profiles

Listeria monocytogenes is a facultative intracellular foodborne pathogen responsible for listeriosis. In a recent study, in which we investigated neglected exogenous routes of transmission of foodborne pathogens into the European Union, we have isolated 15 L. monocytogenes strains in food products, which were imported from the Republic of Moldavia to Romania and illegally sold at a local market.The aim of this study was to characterize the subtype and virulence potential of these 15 L. monocytogenes strains. Multilocus sequence typing revealed that these L. monocytogenes strains belong to six different sequence types (ST2, ST8, ST9, ST20, ST121 and ST155). In addition, in vitro virulence assays using human intestinal epithelial Caco2 and macrophage-like THP1 cells showed a high strain variability regarding the invasion efficiency in Caco2 cells (0.98–2.78%) and the intracellular growth rate in both cell types. Both ST121 strains and the ST9 isolate were unable to invade Caco2 cells, and all ST155 strains showed no proliferation inside macrophages and revealed low cytotoxicity. Furthermore we performed sequence analysis of three main virulence factors: PrfA, internalin A (InlA) and listeriolysin O (LLO). The Romanian food isolates showed a high diversity in the InlA and LLO amino acid sequences, whereas the amino acid sequence of PrfA of all strains was identical. Overall, the amino acid sequences of PrfA, InlA and LLO were identical for strains belonging to the same ST. We detected in total 30 different amino acid substitutions, resulting in seven different InlA variants, two of which have not yet been described. The three strains, which were unable to invade Caco2 cells, harboured a premature stop codon resulting in truncated InlA. Furthermore, we detected four different amino acid substitutions in the LLO sequence, which are present in four variants. The number of LLO mutations correlates negatively with intracellular growth in Caco2 and THP1 cells and subsequently with cytotoxicity.In conclusion, we show that L. monocytogenes isolated from food samples from a Romanian black market show distinct virulence profiles, due to a high diversity in the amino acid sequence of main virulence factors.

DNA-Fragments Are Transcytosed across CaCo-2 Cells by Adsorptive Endocytosis and Vesicular Mediated Transport

Dietary DNA is degraded into shorter DNA-fragments and single nucleosides in the gastrointestinal tract. Dietary DNA is mainly taken up as single nucleosides and bases, but even dietary DNA-fragments of up to a few hundred bp are able to cross the intestinal barrier and enter the blood stream. The molecular mechanisms behind transport of DNA-fragments across the intestine and the effects of this transport on the organism are currently unknown. Here we investigate the transport of DNA-fragments across the intestinal barrier, focusing on transport mechanisms and rates. The human intestinal epithelial cell line CaCo-2 was used as a model. As DNA material a PCR-fragment of 633 bp was used and quantitative real time PCR was used as detection method. DNA-fragments were found to be transported across polarized CaCo-2 cells in the apical to basolateral direction (AB). After 90 min the difference in directionality AB vs. BA was >103 fold. Even undegraded DNA-fragments of 633 bp could be detected in the basolateral receiver compartment at this time point. Transport of DNA-fragments was sensitive to low temperature and inhibition of endosomal acidification. DNA-transport across CaCo-2 cells was not competed out with oligodeoxynucleotides, fucoidan, heparin, heparan sulphate and dextrane sulphate, while linearized plasmid DNA, on the other hand, reduced transcytosis of DNA-fragments by a factor of approximately 2. Our findings therefore suggest that vesicular transport is mediating transcytosis of dietary DNA-fragments across intestinal cells and that DNA binding proteins are involved in this process. If we extrapolate our findings to in vivo conditions it could be hypothesized that this transport mechanism has a function in the immune system.

Regulation of Epithelial Innate Immunity through Hypoxia-mediated Autophagy

The gastrointestinal epithelium comprises the primary barrier between omnipresent luminal antigens and the underlying immune cell repertoire, and is subject to profound metabolic fluctuations, particularly in inflammatory bowel disease (IBD). A role for the hypoxia-inducible transcription factors (HIFs) in orchestrating transcriptional changes to promote barrier function has been well defined. Given the importance of HIF signaling to epithelial homeostasis, we hypothesized that HIF-mediated transcriptional changes contribute to epithelial innate immunity. Promoter microarray screens of HIF-enriched genomic DNA, derived from chromatin immunoprecipitation (ChIP-on-chip), highlighted autophagy as a novel pathway coordinately regulated by HIF-1. A fundamental role for autophagy in IBD pathogenesis has recently been elucidated, defined largely by abrogated bacterial killing (xenophagy). However, little is known about transcriptional regulation of coordinated autophagy gene responses, and how these modulate intestinal epithelial cells (IEC) xenophagy. Human intestinal epithelial Caco-2 and T84 cells were used. Hypoxia was defined as pO2 20 torr and pCO2 35 torr. ChIP-on-chip: ChIP was performed with a polyclonal HIF-1a antibody using sheared chromatin from hypoxic Caco-2 cells. ChIP-enriched and input DNA were Cy-labeled and hybridized to a promoter microarray (Switchgear Genomics). The log2 ratio (input-Cy3/HIF-ChIP-Cy5) was analyzed to identify sequences specific for HIF-1a binding. ChIP-qPCR: ChIP-qPCR was used to quantify HIF binding to candidate loci using primer sets flanking putative HRE sites. Salmonella Typhimurium Invasion and O2 Consumption: Caco-2 cells were infected with late-log cultures of S. Typhimurium 14028 at an multiplicity of infection (MOI) of 100. The SDR OxoDish system (Presens) was used to measure the effect of Salmonella invasion on epithelial O2 consumption in real-time. Gentamicin kill assays: Caco-2 cells were infected with S. Typhimurium and incubated in antibiotic-free medium for 30 min, followed by media supplemented with 50 μg/mL gentamicin (Sigma) for 1h. Viable intracellular bacteria were quantified by colony counts following epithelial lysis in 1% Triton-X-PBS. A cohort of promoters for autophagy genes were specifically enriched for HIF binding by ChIP-chip. Transcript levels of candidate genes were induced by 6hrs under hypoxia or prolyl hydroxylase inhibition in Caco-2 and T84 cells, corroborating association between HIF binding and gene expression. Hypoxic Caco-2 cells displayed 3.5-fold higher LC3/p62-positive puncta by IF than normoxic cells. By immunoblot analysis, LC3-II and p62 protein levels were increased and decreased respectively in hypoxic lysates, indicating hypoxia-induced autophagic turnover. Hypoxia stimulation of autophagy increased Salmonella killing, confirming hypoxic induction of xenophagy. Moreover, invasive Salmonella induced a time- and MOI-dependent depletion of O2 with stabilization of HIF protein. To analyze baseline epithelial autophagy in the absence of HIF signaling in vivo, we measured IEC levels of LC3 and p62 protein in HIF-1b−/− mice. Consistent with a role for HIF, the ratio of LC3-I to LC3-II and total levels of p62 were increased in vil-Cre+/HIF-1bfl/fl mice relative to vil-Cre−/HIF-1bfl/fl controls. Modulation of HIF-mediated autophagy by inflammatory hypoxia and/or bacterial pathogens themselves defines a conserved innate response for host protection, and may represent a novel target for development of therapeutic strategies to modify epithelial autophagic targeting of bacteria.

Luminal Bacterial Lipopolysaccharide Induces Intestinal Epithelial Cell Apoptosis via CD14-Mediated Lipid Secondary Messengers in a TLR4-Independent Manner

We have previously reported that human intestinal epithelial cells released on the basolateral side of culturedmonolayers, arginin-cleavage proteolytic activity upon stimulation by the bacterial motif LPS. Concomitantly, LPS was able to induce a significant increase in permeability of intestinal epithelial monolayers. Further we observed that LPS-induced increased permeability of cultured epithelial cells could be prevented by treatment with a large spectrum serine protease inhibitor. Here, we investigated the effects of pathogenic (EPEC) or commensal (HB101) forms of E. coli on the expression of the proteolytic enzyme mesotrypsin by intestinal epithelial cell lines, and the role of proteolytic activity in E. coliinduced increased permeability. Methods: Human intestinal epithelial cell line Caco-2 was polarized on Transwell chambers for 15 days. Cells were apically stimulated for 5 hours with bacterial suspension of E. coli HB101 and EPEC E2348/69 at 2.108 CFU/well, in the presence or not of the serine protease inhibitor FUT-175 (50μg/ml). Paracellular epithelium permeability was evaluated by dextran-FITC apical to basal passage method. We have evaluated the expression of the mesotrypsin/Trypsin IV gene by qRT-PCR for the mRNA and by western-blot using a rabbit polyclonal antibody against human trypsinogen IV protein. Results:We have observed that EPEC, but not HB101 was able to break the barrier function of epithelial cells in monolayers, increasing the transepithelial passage of dextran-FITC. The large spectrum serine protease inhibitor FUT-175 was able to completely prevent this increased permeability in response to EPEC. In parallel, we observed that the trypsinIV/ Mesotrypsin gene was expressed in Caco-2 cells infected with EPEC, at the mRNA levels. In contrast mRNA expression of trypsinIV/Mesotrypsin was significantly down-regulated when Caco-2 cells were exposed to the commensal form of E. coli: HB101. The trypsinogen IV protein was also detected in supernatants of epithelial cells exposed to EPEC, but not detected in supernatants of Caco-2 cells exposed to HB101. Conclusion: The intestinal epithelium infected by a pathogenic form of E. coli (EPEC), but not by a commensal form (HB101) becomes leaky, allowing the passage of macromolecules. The release of serine protease activity seems to play a major role in the breakage of this epithelial barrier function by pathogenic E. coli. Epithelial expression of trypsin IV/mesotrypsin, which is differentially regulated by pathogenic or commensal forms of E. coli, could play an important role in permeability changes and overall in alerting the immune system to pathogen exposure.

Expression of Claudin-2 in intestinal metaplastic mucosa of Cdx2-transgenic mouse stomach

Objective. Cdx2 is expressed in human intestinal metaplastic mucosa and induces intestinal metaplastic mucosa in Cdx2-transgenic mouse stomach. Claudin-2 is a structural component of tight junctions in the intestine and Cdx2 activates the Claudin-2 promoter in the human intestinal epithelial cell line Caco-2. Our aim is to evaluate the expression of Claudin-2 in intestinal metaplastic mucosa of Cdx2-transgenic mouse stomach. Material and methods. The Claudin-2 expression in the normal gastric mucosa and normal intestinal mucosa of wild type mice and the intestinal metaplastic mucosa of Cdx2-transgenic mice was analyzed by immunohistochemistry, Western blotting and quantitative real-time PCR (qRT-PCR). Results. Claudin-2 was expressed in the base of the glands in intestine and intestinal metaplasia while it was not expressed in the body of stomach. Claudin-2 expression was found in the antrum of stomach, while it was weaker than that in the intestine and the intestinal metaplasia. Claudin-2 was also detected in intestinal metaplasia, colon and ileum by both Western blotting and qRT-PCR while it was not detected in gastric body. Conclusion. These results suggest that Cdx2 plays an important role in the expression of Claudin-2 in vivo.

Intestinal Transport of Free Anthraquinones in Caco-2 Cell Model

AIM: To investigate the transepithelial transport of the six free anthraquinones 1,8-dihydroxyanthraquinone (DQ),chrysophanol (CP),emodin (EN),rhein (RN),aloe-emodin (AE) and citreorosein (CN) in the human intestinal Caco-2 cell monolayer model. METHODS: The monolayers of the human intestinal epithelial cancer cell line Caco-2 were incubated with DQ,CP,EN,RN,AE and CN to model its intestinal absorption and transport,respectively. The determination of the compounds was performed by high-performance liquid chromatography. RESULTS: The apparent permeability coefficients (Papp) in the Caco-2 cell monolayers for DQ,CP,EN,RN,AE and CN were (2.16±0.38)×10-6,(3.00±0.09)×10-7,(1.99±0.94)×10-6,(4.58±0.20)×10-6,(6.24±0.19)×10-6 and (7.34±0.61)×10-6 cm ·s-1 from the apical-to-basolateral direction,respectively,and (3.20±0.10)×10-6,(4.50 ± 0.02)×10-7,(2.22 ± 0.62)×10-6,(4.94 ± 0.39)×10-6,(2.87 ± 0.18)×10-6 and (6.80 ± 0.37)×10-6 cm ·s-1 from the basolateral-to-apical direction,respectively. The Papp values of DQ,EN,RN,AE and CN were about 10 times less than those (6.30 ± 0.26)×10-5 cm ·s-1 of propranolol as a reference standard compound of high permeability and the main mechanism of intestinal absorption by passive diffusion,and about 10 times higher than those ( 1×10-7 cm ·s-1) of atenolol as a reference standard compound of poor permeability and the main mechanism of intestinal absorption by passive diffusion. Whereas,the Papp value of CP was at nearly the same magnitude with those of atenolol. In the presence of iodoacetamide,the Papp of AE were not affected in both apical-to-basolateral and basolateral-to-apical directions. CONCLUSION: The intestinal absorption of all test compounds was passive diffusion as the dominating process in Caco-2 cell monolayer model. DQ,EN,RN,AE and CN will be moderately absorbed compounds and CP was estimated to be a poorly absorbed compound.

A novel function of botulinum toxin-associated proteins: The HA component disrupts intestinal epithelial intercellular junctions to increase toxin absorption

Food-borne BoNTs in the gastrointestinal lumen must cross an epithelial barrier to reach nerve terminals to mediate toxicity. The detailed mechanism by which BoNTs traverse this barrier and enter the circulation remains unclear. Type B BoNT forms two distinct complexes, 12S and 16S, through associations with different sets of non-toxic proteins (non-toxic neurotoxin-associated proteins, NAPs). The 12S toxin is composed of BoNT and a non-toxic protein lacking hemagglutinin (HA) activity, and it is designated non-toxic non-HA (NTNH). The 16S toxin consists of BoNT, NTNH and HA proteins. NAPs are known to markedly increase the oral toxicity of BoNT. We found that type B HA proteins play an important role in the intestinal absorption of BoNT by disrupting the paracellular barrier of the intestinal epithelium. This action facilitates and enhances the transepithelial delivery of BoNT both in vitro and in vivo (T. Matsumura, et al., 2008. Cell. Microbiol. 10, 355–364). Recent experiments showed that type A HA proteins have a similar disrupting activity with greater potency as compared to type B HA proteins in human intestinal epithelial cell lines, Caco-2 and T84. These and other results will be presented and discussed.

Effects of extracellular iron concentration on calcium absorption and relationship between Ca2+and cell apoptosis in Caco-2 cells

To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry. Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase XI and dispase I. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 micromol/L) increased the FI of Caco-2 cells from 27.50+/-13.18 (control, n=150) to 35.71+/-13.99 (n=150, P<0.01), 72.19+/-35.40 (n=150, P<0.01) and 211.34+/-29.03 (n=150, P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 micromol/L). The FI value of Caco-2 cells treated by FAC was 185.85+/-33.77 (n=150, P<0.01), 122.73+/-58.47 (n=150, P<0.01), and 53.29+/-19.82 (n=150, P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187 (0.1, 1.0 and 10 micromol/L) increased the FI of Caco-2 cells from 40.45+/-13.95 (control, n=150) to 45.19+/-21.95 (n=150, P<0.01), 89.87+/-43.29 (n=150, P<0.01) and 104.64+/-51.07 (n=150, P<0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell number was positively correlated with [Ca2+]i. Ca2+ absorbability is increased with the decrease of extracellular iron concentration Fe3+ and hindered with the increase of Fe3+ consistence out of them. Furthermore, increase of [Ca2+]i can induce apoptosis in Caco-2 cells.

Insulin decreases the secretion of apoB-100 from hepatic HepG2 cells but does not decrease the secretion of apoB-48 from intestinal CaCo-2 cells

We compared the acute effect of insulin on the human colonic intestinal epithelial cell line CaCo-2 and the transformed human hepatic cell line HepG2. Over 24 h, 100 nM and 10 microM insulin significantly inhibited the secretion of apolipoprotein (apo) B-100 from HepG2 cells to 63 and 49% of control, respectively. Insulin had no effect on the secretion of apoB-48 from CaCo-2 cells. There was no effect of insulin on the cholesterol ester or free cholesterol concentrations in HepG2 or CaCo-2 cells. HepG2 and CaCo-2 cells bound insulin with high affinity, leading to similar stimulation of insulin receptor protein tyrosine kinase activation. Protein kinase C or mitogen-activated protein kinase activity in the presence or absence of insulin was not correlated with apoB-48 production in CaCo-2 cells. Therefore, insulin acutely decreases the secretion of apoB-100 in hepatic HepG2 cells, but does not acutely modulate the production or secretion of apoB-48 from CaCo-2 intestinal cells.